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Phosphorylation of ERM proteins at filopodia induced by Cdc42
N Nakamura1, N Oshiro, Y Fukata
1Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.
Background:
ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at filopodia and microvilli-like structures. We have shown that Rho-associated kinase (Rho-kinase)/ROKalpha/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273, 34663- 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized.
Results:
Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)-terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C-terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C-terminal threonine in a cell-free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C-terminal threonine-phosphorylated ERM proteins at filopodia induced by Cdc42V12.
Conclusion:
The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.
Insights
The study reveals that Cdc42-induced filopodia formation involves the phosphorylation of ezrin, radixin, and moesin (ERM) proteins. Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is identified as a key kinase responsible for this ERM phosphorylation at filopodia.
Area of Science:
- Cell biology
- Molecular biology
- Cytoskeleton dynamics
Background:
- Ezrin, radixin, and moesin (ERM) proteins link the cell membrane to the cytoskeleton.
- ERM proteins are crucial for microvilli-like structure formation via Rho-kinase phosphorylation.
- The role of ERM proteins in filopodia formation remained less characterized.
Purpose of the Study:
- To investigate the role of ERM protein phosphorylation in Cdc42-induced filopodia formation.
- To identify the kinase responsible for ERM phosphorylation during filopodia development.
Main Methods:
- Utilized NIH 3T3 cells and transfection with constitutively active Cdc42 (Cdc42V12).
- Employed an antibody specific for C-terminal threonine-phosphorylated ERM proteins.
- Performed cell-free kinase assays and co-expression of dominant-negative Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK).
Main Results:
- Cdc42V12 expression induced filopodia formation and ERM phosphorylation at the filopodia tips.
- Phosphorylated ERM levels were lower and diffuse in control cells.
- Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) phosphorylated moesin at the C-terminus in vitro.
- Inhibition of MRCK blocked Cdc42-induced filopodia formation and ERM phosphorylation at filopodia.
Conclusions:
- Cdc42-induced filopodia formation is associated with ERM protein phosphorylation.
- MRCK is a strong candidate kinase for phosphorylating ERM proteins at filopodia.